Multimodal single cell profiling of bone fracture microenvironment

Age-specific alterations of the adaptive immune system include low-grade chronic inflammation, which contributes to the unfavorable outcome of the bone healing process in the elderly. In this work, we aimed to characterize the effect of immune experience and age on cellular phenotype during the early inflammatory phase after bone fracture by using a mouse model which included young, aged, and immune-aged mice 2- and 5-days post osteotomy. Single-cell proteo-genomics quantified thousands of transcriptomes of cells isolated from three different bone regions (fracture hematoma, proximal and distal bone marrow). The fracture hematoma is a very distinct tissue consisting of coagulated blood caused by rupture of adjacent blood vessels, bone marrow influx and cells attracted from the surrounding tissue. The hematoma matures with the healing process and the adjacent bone marrow (proximal and distal to the fracture gap) supports the healing process. We compared young, aged (non-experienced immune system) and immune-aged (experienced immune system) animals during the immediate-to-early response to bone fracture, whereby mice (three mice per group) were sacrificed 2- or 5-days post osteotomy. We dissected 3 anatomical regions from each bone (fracture hematoma, adjacent proximal and distal bone marrow). After cell dissociation, enrichment sorting and labeling, cell encapsulation and library preparation were performed, followed by single-cell proteo-genomics, which included transcriptome quantifications and surface protein detection using the CITE-seq approach.